This invention relates generally to the area of diagnosis of heart disease, and specifically relates to methods of diagnosis of heart failure, cardiac ischemia, or hypoxia by detecting the level, e.g., concentration, of a non-polypeptidic cardiac marker as an indicator of heart damage, particularly chronic underlying coronary artery disease, and for monitoring of therapeutic regimes designed to alleviate cardiac ischemia or hypoxia.
Ischemic heart disease is the major form of heart failure. Heart failure affects millions of people worldwide and is the leading cause of death in the United States. The most common manifestation of cardiac ischemia is chest pain (angina pectoris) which can lead to heart attack (acute myocardial infarction or AMI) and sudden death. In addition to those who exhibit clinical symptoms of ischemic heart disease, many other individuals are at high risk of developing heart disease based on indicators such as hypertension conditions, high levels of serum cholesterol and/or family history.
Myocardial ischemic disorders occur when cardiac blood flow is restricted (ischemia) and/or when the oxygen supply to heart muscle is compromised (hypoxia) such that the heart""s demand for oxygen is not met. Atherosclerosis of the coronary artery is the most common cause of ischemia-associated symptoms such as angina pectoris. Ischemia and hypoxia can be transient and reversible, but can also lead to infarction. During infarction, cardiac tissue is damaged and the heart cells become permeabilized, releasing a portion of their contents to the surrounding milieu, including cardiac enzymes and other biochemical markers. These cellular markers, such as creatine kinase (CK), lactic acid dehydrogenase (LDH) enzymatic activities and creatine kinase-MB (CKMB) and troponin (I and T) and myoglobin mass levels, are then detectable in the serum.
Current diagnostic procedures generally assess the extent of cardiac tissue damage after clinical signs have appeared. At that point, however, the disease may have progressed to an extent where AMI is imminent or has already occurred. Current methods of identifying and confirming infarction require more time than is often available in emergency situations where rapid evaluation is critical for effective patient treatment and survival. Moreover, about 25% of AMI patients display atypical symptoms and many known tests result in false negatives, resulting in the unintentional discharge of about 5% of patients who have AMI (Mair J. et al., Clin. Chem. 41:1266-1272, 1995; Newby L. K. et al., Clin. Chem. 41:1263-1265, 1995). In an emergency medical facility, electrocardiography (ECG) monitoring of suspected AMI patients is the most rapid diagnostic method for detecting AMI, although it successfully detects only about half of AMI patients (Mair et al., 1995).
Electrocardiography and currently available diagnostic blood tests are generally not effective for early detection of myocardial ischemia that precedes the damage associated with AMI because the tests detect infarction-associated tissue damage. They are not effective in early detection of chronic underlying coronary artery disease and the resulting myocardial ischemia that precedes the damage associated with AMI. Currently, the only diagnostic for chronic underlying coronary artery disease is ECG monitoring during exercise stress (e.g., treadmill exercise) is generally used to confirm the clinical symptoms of angina. Such stress testing is usually given after the patient has experienced symptoms and sought treatment (e.g., at an emergency medical facility). Although stress testing is sometimes used to screen asymptomatic patients, testing is costly, time-consuming and generally not amenable to routine screening of large numbers of patients. Furthermore, exercise stress test evaluations result in about 15% false negatives.
Diagnostics tests have been developed that use cardiac proteins to determine whether or not the source of the patient""s chest pain is cardiac and if so, whether the patient has suffered a myocardial infarct or is suffering from unstable angina (see, e.g., U.S. Pat. Nos. 5,290,678, 5,604,105, and 5,710,008). These tests do not give an early warning for when myocardial infarct is forthcoming. Thus, a non-invasive, sensitive, and reliable point-of-care xe2x80x98bedside testxe2x80x99 is needed for the early detection of cardiac ischemia, particularly for people at risk for heart disease.
In view of the need for rapid and reliable methods for detecting cardiac ischemia in the absence of symptoms, particularly for screening those at high risk of heart disease, the present invention is an early detection assay for cardiac ischemia or hypoxia.
The present invention provides diagnostic methods for the early detection of heart disease (e.g., heart failure, cardiac ischemia, and cardiac hypoxia) in mammals, particularly humans, by monitoring serum or whole blood levels of non-polypeptidic cardiac markers, e.g., sphingosine and/or its metabolites. For instance, an early event in the course of cardiac ischemia (i.e., lack of blood supply to the heart) is an excess production by the heart. muscle of certain naturally occurring non-polypeptidic compounds, or cardiac markers, such as, but not limited to, sphingosine (SPH; D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol or sphingenine), its isomers, and metabolites; ceramide (Cer, n-acylsphingosine), sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC, lysosphingomyelin), and glycosphingolipids and lysophospholipids such as lysophosphatidic acid (LPA), and the metabolites of any of the foregoing. The present invention is based on the observation that SPH is increased in the serum and suggests that blood sphingolipid levels represent a new biochemical marker for cardiac ischemia.
Evidence indicates that the cardiac source of tumor necrosis factor alpha (TNFxcex1) may be responsible for the characteristic increased serum sphingolipids resulting from cardiac ischemia. Accordingly, preferred embodiments of the invention provide that serum SPH levels, or levels of other related lipids having a sphingosine backbone, be used in combination with levels of a secondary marker, e.g., serum TNFxcex1, as an index of ischemia. Of course, other non-polypeptidic cardiac markers can also be used in conjunction with a secondary marker such as TNFxcex1 to calculate such an index. This dual analyte measure is referred to as Myocardial Risk Factor (MRF).
Kits according to the invention provide cost-effective and rapid tests that can be used to identify and predict, among other cardiac conditions, acute myocardial infarction (AMI) and to confirm that angina pectoris results from cardiac ischemia. In addition, the present invention can be used for simple screenings of early ischemic or hypoxic events before symptoms are presented, e.g., in persons with high risk for heart disease and for persons experiencing other forms of heart failure, including myocarditis, the cardiomyopathies, and congestive and idopathic heart failure. Moreover, the methods and compositions according to the invention can be used to monitor the effectiveness of therapeutic interventions designed to relieve the ischemia and heart failure.
Thus, in one aspect, the invention provides a method of detecting heart disease characterized by cardiac ischeria or hypoxia in a mammal comprising the steps of (a) measuring a level of a non-polypeptidic cardiac marker in the test sample from the mammal; and (b) determining if the level of the cardiac marker measured in the test sample correlates with cardiac ischemia or hypoxia.
xe2x80x9cIschemiaxe2x80x9d means a condition where the cardiac muscle receives insufficient blood supply, whereas xe2x80x9chypoxiaxe2x80x9d means a condition where the cardiac muscle receives insufficient oxygen.
The term xe2x80x9cmammalxe2x80x9d refers to such organisms as mice, rats, rabbits, goats, horse, sheep, cattle, cats, dogs, pigs, more preferably monkeys and apes, and most preferably humans.
In preferred embodiments, the subject of the methods of the invention is a human, and the test sample used is preferably a body fluid. The body fluid is preferably selected from the group consisting of blood, urine, lymph, and saliva, although any other body fluid, such as serum, gastric juices, and bile, may be used. Most preferably the body fluid is blood.
The term xe2x80x9cnon-polypeptidic cardiac markerxe2x80x9d means a compound that is not considered to be a peptide by those skilled in the art, even though it may contain a peptide bond or an amide bond, and is uniquely associated with the heart, such that the heart and cardiac functions are the source of the compound.
The non-polypeptidic cardiac marker is preferably a lipid and more preferably a sphingolipid. A xe2x80x9clipidxe2x80x9d means a substance that is insoluble in water that can be extracted from cells by organic solvents of low polarity. Lipids include compounds such as terpenes, steroids, fats, and fatty acids. A xe2x80x9csphingolipidxe2x80x9d means a compound that shares the sphingosine backbone containing an 18-carbon chain amino alcohol of the general formula CH3(CH2)14CH(OH)CH(NH2)CH2xe2x80x94R, where R may be any organic substituent. xe2x80x9cSphingosinexe2x80x9d means the compound of formula CH3(CH2)14CH(OH)CH(NH3+)CH2OH, as shown in FIG. 1. The scope of the invention also includes compounds where the carbon chain of the sphingolipid contains centers of unsaturation (i.e., double bonds or triple bonds), or where hydroxide or the amine substituents are further substituted with organic substituents. It is also understood xe2x80x9csphingolipidxe2x80x9d refers to any isomer, e.g., threo-sphingosine, erythro-sphingosine, and L and D isomers of a sphingolipid, as well as any metabolite of any of the foregoing non-polypeptidic cardiac markers.
The non-polypeptidic cardiac marker is more preferably sphingosine or one of its metabolites. The metabolite is preferably selected from the group consisting of ceramide (Cer, n-acylsphingosine), sphingosine-1-phosphate (S1P), sphingosylphosphorylcholine (SPC), and dihydrosphingosine (DHSPH). The structures of these metabolites are shown in FIG. 1.
In preferred embodiments, the measuring step of the methods of the invention comprises measuring the marker level by a method selected from the group consisting of chromatography, immunoassay, enzymatic assay, and spectroscopy, where the cardiac marker is directly or indirectly detected. xe2x80x9cMarker levelxe2x80x9d means the amount of the marker in the sample or in the mammal, and refers to units of concentration, mass, moles, volume, preferably concentration, or other measure indicating the amount of marker present in the sample.
The chromatographic method is preferably high performance liquid chromatography (HPLC) or gas chromatography (GC). The spectroscopic method is preferably selected from the group consisting of ultraviolet spectroscopy ((UV or UV/V is spectroscopy), infrared spectroscopy (IR), and nuclear magnetic resonance spectroscopy (NMR).
The immunoassay preferably detects a non-polypeptidic cardiac marker selected from the group consisting of Cer, SPH, S1P, DHSPH, and SPC. Preferably, the immunoassay detects the non-polypeptidic cardiac marker in the test sample using anti-marker antibodies.
The term xe2x80x9cantibodyxe2x80x9d refers to a monoclonal or polyclonal antibody or antibody fragment having specific binding affinity to a non-polypeptidic cardiac marker.
By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody or antibody fragment binds to target compounds with greater affinity than it binds to other compounds under specified conditions. Antibodies or antibody fragments having specific binding affinity to a compound may be used in methods for detecting the presence and/or amount of the compound in a sample by contacting the sample with the antibody or antibody fragment under conditions such that an immunocomplex forms and detecting the presence and/or amount of the compound conjugated to the antibody or antibody fragment.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110.
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the target compound. The term xe2x80x9cantibody fragmentxe2x80x9d also includes single change antibodies.
In preferred embodiments, the determination step of the method of invention is a comparison between the concentration of the cardiac marker and a predetermined value for the marker. In preferred embodiments, the predetermined value is indicative of a normal cardiac condition. This predetermined value can be determined using the methods of the present invention as described in Detailed Description of the Invention, below, and can be specific for a particular patient or generic for a given population. The predetermined value is preferably obtained from a mammal in the same species and approximately the same age as the mammal providing the test sample. In certain embodiments, the predetermined value may have been established by prior measurement of the particular patient""s marker levels when the patient was healthy.
In practicing the methods of the invention, the level.(e.g., concentration) of the non-polypeptidic cardiac marker in the test sample is preferably higher than a predetermined value for that marker, which higher level correlates with or indicates ischemia, hypoxia, or another form of heart failure. However, with certain non-polypeptidic cardiac markers, the level of the marker in the test sample may be lower than the predetermined value in order to indicate ischemia, hypoxia, or another form of heart failure.
In a further aspect, the invention relates to a method of detecting heart failure (e.g., cardiac ischemia or hypoxia) in a mammal comprising the steps of (a) measuring a level of one or more non-polypeptidic cardiac markers in a test sample from the mammal; (b) measuring a level of one or more secondary cardiac markers in the test sample; and (copyright) determining if the level of the cardiac markers measured in the test sample correlates with cardiac ischemia or hypoxia The secondary cardiac marker(s) is(are) preferably a pro-inflammatory cytokine such as interleukin (IL-1, 2, or 6), interferon gamma (IFNxcex3), and particularly tumor necrosis factor alpha (TNFxcex1). TNFxcex1 has been implicated in the pathophysiology of ischemia and hypoxia. As those in the art will appreciate, the instant methods and compositions may also include measurement of the levels of two (or) more non-polypeptidic cardiac markers, alone or in conjunction with one or more secondary cardiac markers. For purposes of this invention, a xe2x80x9csecondaryxe2x80x9d cardiac marker is an intercellular or intracellular messenger which precipitates or contributes to the underlying cause of heart failure. In other embodiments of this aspect of the invention, the level of one or more xe2x80x9ctertiaryxe2x80x9d cardiac markers can also be determined and used in conjunction with levels determined for the non-polypeptidic cardiac markers(s), or non-polypeptidic and secondary cardiac marker(s) tested. For purposes of this invention, a xe2x80x9ctertiaryxe2x80x9d marker is one associated with disruption of cardiac cells, and generally relates to proteins, polypeptides, and nucleic acids released from ruptured or lyzed cardiac cells. Certain preferred examples of such markers include CK, LDH, CKMB, and troponin. Other preferred examples of such tertiary cardiac markers include nucleic acids specific for cardiac cells, particularly mRNA, expressed predominantly, and preferably only in cardiac cells.
In another aspect, the method of the invention concerns calculating a myocardial risk factor (MRF). As used herein, the MRF has a mathematical relation with the measured level, preferably concentration, of at least one non-polypeptidic cardiac marker and the measured level, preferably concentration, of a second cardiac marker, e.g.,. TNFxcex1. The mathematical relation is preferably a product of the measured level (e.g., concentration) of at least one non-polypeptidic cardiac marker, preferably a sphingolipid, and the measured level (e.g., concentration) of the second marker, preferably TNFxcex1. Of course, other mathematical relationships between different markers are also within the scope of the invention. For example, such relationship may involve two non-polypeptidic cardiac markers, a non-polypeptidic cardiac marker, a secondary cardiac marker, and a tertiary cardiac marker, or a non-polypeptidic cardiac marker and a tertiary marker.
In another aspect, the invention provides for a method of preventing or reducing the severity of a subsequent acute myocardial infarction (or other form of heart failure) by detecting cardiac ischemia or hypoxia, as described herein, and taking a preventive measure. The preventive measure is preferably selected from the group consisting of coronary bypass surgery, preventive angioplasty, and/or administering therapeutically effective amounts of one or more anticoagulants, thrombolytics, or other pharmaceutical products intended to alleviate the ischemic or hypoxic condition.
Furthermore, the methods of the invention allow a health care professional to determine the prognosis of a patient following a cardiac procedure by detecting cardiac ischemia or hypoxia. The cardiac procedure is preferably selected from the group consisting of coronary bypass surgery, preventive angioplasty, and administering one or more anticoagulant, although other cardiac procedures are also within the scope of the present invention.
In another aspect, the invention provides for kits for detecting heart failure, such as may result from cardiac ischemia or hypoxia, in a mammal. Preferably, such kits comprise a composition for detecting an abnormal level of at least one non-polypeptidic cardiac marker in a test sample obtained from a mammal. Preferably, the composition enables measuring the abnormal level in a quantitative manner, although measuring the abnormal level can also be accomplished in a semi-quantitative manner (e.g., is the level above or below a pre-determined threshold value). The composition may preferably comprise a substrate, which may preferably be an antibody which binds to a non-polypeptidic cardiac marker selected from the group consisting of Cer, SPH, S1P, DHSPH, and SPC. The composition may also include one or more other substrates, e.g., an anti-TNFxcex1 antibody, to detect other cardiac-specific markers. The substrate may be affixed to a solid support for easy handling. Common forms of solid support include, but are not limited to, plates, tubes, and beads, all of which could be made of glass or another suitable material, e.g., polystyrene, nylon, cellulose acetate, nitrocellulose, and other polymers. The solid support can be in the form of a dipstick, flow-through device, or other suitable configuration.
In a xe2x80x9cquantitativexe2x80x9d measurement, the step of measuring results in the production of a value which accurately shows the level of the cardiac marker in the test sample. In a xe2x80x9csemi-quantitativexe2x80x9d measurement, the step of measuring results in the indication of whether the level of the cardiac marker is within a particular range. Semi-quantitative methods include, for example, but are not limited to, color indicators or depiction of certain symbols, where each color or symbol represents a concentration range.
Preferably, the level of the cardiac marker(s) detected in the practice of this invention is(are) different than a standard or reference measure that indicates a normal cardiac condition. More preferably, the level of the cardiac marker detected is greater than the standard measure.
In preferred embodiments, the level of the cardiac marker(s) measured in accordance with the invention are detected using. a xe2x80x9cnon-invasivexe2x80x9d method, i.e., one which does not require piercing the skin of the subject mammal to obtain the test sample. Non-invasive methods include, but are not limited to, testing body fluids such as saliva, urine, and sweat, or using imaging techniques.
Preferably, the level of the cardiac marker is measured using a kit of the invention by a method selected from the group consisting of chromatography, immunoassay, enzymatic assay, and spectroscopy, where the marker is directly or indirectly detected. The chromatographic method is preferably high performance liquid chromatography (HPLC) or gas chromatography (GC). The spectroscopic method is preferably selected from the group consisting of ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. With regard to the non-polypeptidic cardiac marker, the immunoassay preferably detects Cer, SPH, S1P, DHSPH, or SPC.
In another aspect, the invention provides devices for detecting cardiac ischemia or hypoxia in a mammal, where the device informs the user of an abnormal level of at least one non-polypeptidic cardiac marker in a test sample obtained from a mammal.
The informing step preferably includes the step of detecting said cardiac marker, which, in turn, is preferably performed by a non-invasive procedure. The informing step also preferably comprises the step of comparing the level of the marker with a predetermined value. Finally, the informing step preferably includes a step of alerting a user, who may or may not be the wearer of the device, as to the level of the marker. The device may display the level of the marker, sound an alarm when the level of the maker surpasses a pre-determined threshold, or inform emergency personnel, such as police, ambulance, or fire department.
The mammal for whom the device is used is preferably a human. The device preferably tests a body fluid for the presence of a non-polypeptidic cardiac marker, which preferably is a sphingolipid, for example, sphingosine or a metabolite thereof. The sphingosine metabolite is preferably selected from the group consisting of Cer, S1P, SPC, and DHSPH.
Yet another aspect of the invention concerns compositions for detecting an abnormal level (e.g., concentration) of at least one non-polypeptidic cardiac marker in a test sample (preferably a body fluid) obtained from a mammal, particularly a human. In certain embodiments, the level of the non-polypeptidic marker is measured quantitatively; in other embodiments, the measurement is semi-quantitative.
In preferred embodiments of this aspect, the composition comprises an antibody, anti-body fragment, or antigen binding domain of an antibody, that specifically binds a non-polypeptidic cardiac marker. In embodiments employing an antibody, the antibody can be a polyclonal, and preferably a monoclonal antibody. In certain embodiments, the non-polypeptidic cardiac marker detected by the composition is a lipid, preferably a sphingolipid or a metabolite thereof, particularly Cer, SPH, S1P DHSPH, and SPC.
Compositions according to the invention may also comprise, in addition to a moiety capable of detecting a non-polypeptidic cardiac marker, a second moiety capable of detecting a secondary cardiac marker (e.g., TNFxcex1, IL-1, IL-2, IL-6, and IFNxcex3), and/or a third moiety capable of detecting a tertiary cardiac marker (e.g., CK, CKMB, LPH, a troponin, and a nucleic acid, particularly a nucleic acid specific to cardiac cells). When the tertiary cardiac marker comprises a nucleic acid probe substantially complementary to at least a sufficient portion of the nucleotide sequence of the nucleic acid so as to enable selective hybridization between the probe and nucleic acid stringent conditions.
In preferred embodiments, the compositions of the invention further comprise a solid support to which the moiety detecting the cardiac marker(s) is or can be attached. In certain embodiments, attachment of the detecting moiety, e.g. an antibody or nucleic acid probe, is via a covalent linkage with the solid support. In other embodiments, attachment may be via a non-covalent linkage, for example, between members of a high affinity binding pair. Many examples of high affinity binding pairs are known in the art, and include biotin/avidin, ligand/receptor, and antigen/antibody pairs.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description, and from the claims.